Starch is a polymeric carbohydrate of very high molecular weight. Its monomeric units, termed anhydroglucose units, are derived from dextrose, and the complete hydrolysis of starch yields dextrose. In the United States, dextrose is manufactured from corn starch; in Europe from corn starch and potato starch; and in Japan from corn starch and white sweet potato starch.
Until 1960, dextrose was prepared from starch by acid hydrolysis. The method of preparation involved heating starch with hydrochloric or sulfuric acid at temperatures of 120.degree.-145.degree. C., then neutralizing the hydrolysis mixture with sodium carbonate, clarifying, and crystallizing the dextrose. Unfortunately, the yield of dextrose is lowered by the formation of relatively large amounts of reversion products, i.e., products which are formed by the recombination of dextrose molecules. Also, because of the high temperature and low pH of the hydrolysis reaction, some of the dextrose produced is converted to hydroxymethylfurfural, levulinic acid and color bodies. The formation of such degradation products is irreversible and, to the extent they are formed, the yield of desired dextrose is, of course, adversely affected. Still further, the use of hydrochloric acid or in some instances, sulfuric acid, and the subsequent neutralization of this acid with alkali results in the formation of inorganic salts which interfere with crystallization of the final dextrose product.
Later, hydrolysis of starch to dextrose was accomplished by means of enzymes. The principal enzyme used for such purposes was, and continues to be, glucoamylase. This enzyme effectively hydrolyzes the starch by cleaving one molecule of dextrose at a time from the starch molecule. As a practical matter, however, it is necessary first to thin the starch before subjecting it to the action of glucoamylase. This thinning step may be accomplished either by means of acid or enzyme. The starch is thinned to a D.E. of about 10-20, then treated with glucoamylase. This two-stage process is referred to as an acid-enzyme process or an enzyme-enzyme process, depending upon the nature of the thinning step employed.
In the acid-enzyme process, starch is liquefied and hydrolyzed in an aqueous suspension containing 20 to 40 percent starch and an acid, such as hydrochloric acid. The suspension is then heated to a high temperature, i.e., a temperature between about 70.degree. C. and about 160.degree. C. and at a pH between about 1 and 4.5 to liquefy and partially hydrolyze the starch. Typical acid-enzyme processes are disclosed in U.S. Pat. Nos. 2,305,168; 2,531,999; 2,893,921; 3,021,944; and 3,042,584.
In the enzyme-enzyme process, starch is liquefied and partially hydrolyzed in an aqueous suspension containing 20 to 40 percent starch and a liquefying enzyme, such as bacterial .alpha.-amylase enzyme at a temperature of from about 85.degree. C. to about 105.degree. C. The dextrose equivalent of the liquefied and partially hydrolyzed starch is generally less than about 20 and preferably less than about 10. The mixture is then subjected to a temperature above about 95.degree. C. and preferably between 110.degree. C. and 150.degree. C. to insure complete starch solution. The starch hydrolyzate is then cooled to a temperature of less than 95.degree. C. and subjected to further treatment with bacterial .alpha.-amylase to hydrolyze the starch to a D.E. of up to about 20. This process is disclosed and claimed in U.S. Pat. No. 3,853,706.
By either process the digested starch may thereafter be converted to dextrose or dextrose-containing syrups by other enzymes such as glucoamylase. Glucoamylase preparations are produced from certain fungi strains such as those of the genus Aspergillus; for example, Aspergillus phoenicis, Aspergillus niger, Aspergillus awamori, and certain strains from the Rhizopus species and certain Endomyces species. Glucoamylase effects the hydrolysis of starch proceeding from the non-reducing end of the starch molecule to split off single glucose units at the alpha-1,4 linkages or at the alpha-1,6 branch points. Commercial glucoamylase enzyme preparations comprise several enzymes in addition to the predominating glucoamylase; for example, small amounts of proteases, cellulases, .alpha.-amylases, and transglucosidases.
Considerable interest has developed in the use of immobilized enzyme technology for production of dextrose from starch. Immobilization may increase enzyme stability, the enzyme material may be re-used repeatedly, and a more precise control of the reaction is possible. Various procedures have been described for the immobilization of glucoamylase.
References which review the art of enzyme immobilization, with particular attention to the immobilization of glucoamylase are given in U.S. Pat. No. 4,011,137.
Processes for the immobilization of enzymes on colloidal silica have recently been reported. U.S. Pat. No. 3,802,997 disclosed that "colloidal silica could be used to bind enzymes." However, this patent taught that it is necessary to combine a substrate of the enzyme with the carrier before the enzyme is bound to the carrier. The process included an expensive freeze drying or spray drying step to isolate the product. In U.S. Pat. No. 3,796,634, colloidal silica was also used as a carrier for enzymes. In this case, glutaraldehyde was used to cross-link the enzyme and chemically bind it to the silica. Alternatively, polyethyleneimine was first bound to the silica and then the enzyme was attached to the polyamine on the silica surface by means of a cross-linking agent. Very finely divided particles were obtained by the methods described in both of these patents. Such small enzyme-containing particles are difficult to remove from batch reactions and produce unmanageable pressure drops when attempts are made to use them in column operation.
In a pending U.S. application, Ser. No. 780,374, filed Mar. 23, 1977 now U.S. Pat. No. 4,144,127, a glucose isomerase enzyme was bound to colloidal silica. Binding of the enzyme was improved by the use of glutaraldehyde and the particle size was increased by freezing the mixture and thawing it at least two times. This produced granules or flakes having a particle size of 20-100 mesh which were large enough for good flow in a column isomerization reaction. No examples of the binding of glucoamylase were given in any of these patents.
It has been discovered that the small particles of cationic colloidal silica can be agglomerated to larger particles if they are gelled by raising the pH to about 6.5, then frozen and thawed. It has been discovered furthermore, that when the colloidal silica is agglomerated by this method in the presence of glucoamylase, the enzyme is immobilized in an active form. No cross-linking agent is necessary to bind the enzyme to the silica, and the enzyme does not leach out when the particles are suspended in or washed with aqueous solutions.
In contrast, if the colloidal silica is first agglomerated and the resulting particles are then contacted with the enzyme, very little of the enzyme is bound to the silica.
An important advantage of this enzyme composite is that it is relatively noncompressible. For example, it undergoes no volume change when spun in a centrifuge at 2000 rpm for 15 minutes. This noncompressibility, coupled with good particle size makes the enzyme composite very suitable for carrying out enzymatic conversions in fixed bed reactors with a minimum of pressure drop across the reaction zone. These properties of the composite also make it useful for enzymatic conversions in expanded bed (upflow) reactors, continuous stirred tank reactors and batch reactors.